The poxviridae comprise a large family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells. The family of poxviridae can be divided into the subfamily chordopoxvirinae (vertebrate poxviruses) and extomopoxvirinae (insect poxviruses).
The chordopoxvirinae comprise several animal poxviruses (classified in different genera) of significant economical importance, such as camelpox viruses, sheeppox virus, goatpox virus or avipoxviruses, in particular fowlpoxvirus. For the vaccination of livestock against sheeppox and goatpox attenuated live-virus and inactivated vaccines are available. For the vaccination of poultry recombinant vaccines have been developed using fowlpox virus as a vector.
Since fowlpoxvirus infects human cells it is assumed that it can also be used as a vector to express heterologous genes in humans and to induce a corresponding immune response. Fowlpoxviruses containing HIV genes in the genome are disclosed in U.S. Pat. Nos. 5,736,368 and 6,051,410.
In humans the variola virus, a member of the genus Orthopoxvirus, was by far the most important poxvirus. Vaccinia virus, also a member of the genus Orthopoxvirus in the family of poxviridae, was used as live vaccine to immunize against smallpox. Successful worldwide vaccination with Vaccinia virus culminated in the eradication of variola virus (The global eradication of smallpox. Final report of the global commission for the certification of smallpox eradication; History of Public Health, No. 4, Geneva: World Health Organization, 1980). Since that WHO declaration, vaccination has been discontinued for many years except for people at high risk of poxvirus infections (e.g. laboratory workers). Vaccination programs are again becoming of interest in view of the risk that variola virus is used in biological warfare or by bioterrorists.
More recently, Vaccinia viruses have also been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M., Smith, G. L. and Moss, B. [1982] P.N.A.S. USA 79, 7415–7419; Smith, G. L., Mackett, M. and Moss, B. [1984] Biotechnology and Genetic Engineering Reviews 2, 383–407). This entails DNA sequences (genes), which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the Vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant Vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83, 286 and No. 110, 385). The recombinant Vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
The use of Vaccinia virus as vector for the development of recombinant live vaccines has been affected by safety concerns and regulations. Most of the recombinant Vaccinia viruses described in the literature are based on the Western Reserve strain of Vaccinia virus. It is known that this strain has a high neurovirulence and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65–70 [1987]). On the other hand the Modified Vaccinia virus Ankara (MVA) is known to be exceptionally safe. MVA has been generated by longterm serial passages of the Ankara strain of Vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. [1975] Infection 3, 6–14; Swiss Patent No. 568, 392). Examples for MVA virus strains deposited in compliance with the requirements of the Budapest Treaty are strains MVA 572, MVA 575 and MVA-BN deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury (UK) with the deposition numbers ECACC V94012707, ECACC V00120707 and ECACC V00083008, respectively. MVA is distinguished by its great attenuation that is to say by diminished virulence or infectiosity while maintaining good immunogenicity. The MVA virus has been analyzed to determine alterations in the genome relative to the wild type CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H., Sutter, G. and Mayr A. [1991] J. Gen. Virol. 72, 1031–1038). The resulting MVA virus became severely host cell restricted to avian cells. Furthermore, MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375–390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386–2392 [1974]). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine. Recombinant MVA useful as vaccines have already been constructed and used in clinical trials. WO 98/13500 discloses a recombinant MVA containing and capable of expressing one or more DNA sequences encoding dengue virus antigens. The foreign DNA sequences were recombined into the viral DNA at the site of a naturally occurring deletion in the MVA genome.
Before using poxviruses or recombinant poxvirus for vaccination it is necessary to purify the virus to a certain extent in order to meet regulatory requirements. The traditional way to purify poxviruses, in particular MVA and recombinant MVA is as follows: in a first step cells susceptible to infection with the respective poxvirus are cultivated. In case of MVA the susceptible cells are i.a. chicken embryo fibroblasts. The susceptible cells are infected with the poxvirus and cultivated for a time period sufficient to allow the generation of virus progeny. The cells then are frozen and thawed in order to detach the cells from the culture vial surface and to partially disrupt the cells. The mixture of intact and disrupted cells is spun down. Ultrasound is used to produce a homogenate. Virus is purified from the homogenate by sucrose cushion centrifugation (Joklik WK. “The purification of four strains of poxvirus” Virology 1962; 18:9–18). The key step in this process is the homogenization by using ultrasound (Hedström, K. G. and Lindberg, U., Z. Immun. Forsch. 1969 137:421–430; Stickl, H., Korb, W. and Hochstein-Mintzel, V., Zbl. Bakt., I. Abt. Orig. (1970), 215, 38–50). In industrial processes it is preferred that all process steps are easy to control and reproducible. However the disadvantage in using ultrasound to homogenize the virus-cell suspension is that the ultrasound step is difficult to reproduce in an identical manner, difficult to adjust and it is difficult to scale up the process from laboratory to industrial scale.